Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage

Our plain-English summary of Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage, by S.J. Kim, P. Cheresh, R.P. Jablonski, et al (Free Radical Biology & Medicine, 2016).

The study

• Authors: S.J. Kim, P. Cheresh, R.P. Jablonski, L. Morales-Nebreda, Y. Cheng, E. Hogan, A. Yeldandi, M. Chi, R. Piseaux, K. Ridge, C. Michael Hart, N. Chandel, G.R. Scott Budinger, D.W. Kamp
• Journal: Free Radical Biology & Medicine
• Published: 2016;101:482-490
• Cited by: 77 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Rationale: Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung.

Objective: To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis.

Methods: Crocidolite asbestos (100µg/50µL), TiO2 (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay.

Results: Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced.

Conclusions: Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role for AEC mitochondrial H2O2-induced mtDNA damage in promoting lung fibrosis. We reason that strategies aimed at limiting AEC mtDNA damage arising from excess mitochondrial H2O2 production may be a novel therapeutic target for mitigating pulmonary fibrosis.

Published by Elsevier Inc.

In plain English

This laboratory study used mice and lung cells grown in dishes. The authors compared ordinary (wild-type) mice with MCAT mice, genetically altered to make extra catalase (an enzyme converting hydrogen peroxide to water) inside their mitochondria. Crocidolite asbestos, titanium dioxide (a comparison particle), bleomycin (another fibrosis-causing substance) or a salt-solution control was put into the windpipe; lungs were examined 21 days later. Asbestos-induced lung fibrosis was significantly lower in MCAT mice than in wild-type mice, and MCAT mice were protected against bleomycin. After asbestos, dying cells were 3% of airway junction cells counted in MCAT mice versus 10% in wild-type mice, and MCAT type II cells had less mitochondrial DNA damage. Dying type II cells were 3% (MCAT) versus 4% (wild-type) of junction cells counted, not a statistically significant difference. In dish experiments with amosite asbestos, type II cells from MCAT mice produced less mitochondrial reactive oxygen. The authors concluded that their findings suggest a role for mitochondrial hydrogen peroxide-induced damage to the mitochondrial DNA of alveolar epithelial (air-sac lining) cells in promoting lung fibrosis. They noted they had not excluded roles for other cell types.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment in genetically modified and wild-type mice, with additional cell-culture (in vitro) experiments
• Who was studied: Male and female 8- to 10-week-old wild-type C57Bl/6J mice and MCAT mice (which overexpress human catalase targeted to mitochondria). The authors state 6 animals per group unless noted; figure legends give group sizes of 4 to 9. Also studied: primary alveolar type II cells isolated from these mice, and MLE-12 lung epithelial cells. The work was done at Northwestern University and the Jesse Brown VA Medical Center, Chicago.
• Exposure: In mice: a single instillation into the windpipe of 100 µg crocidolite asbestos or titanium dioxide (negative control particle) in 50 µL PBS, or 0.025 units bleomycin or saline in 50 µL, with lungs harvested at 21 days. In cells: amosite asbestos (25 µg/cm2) or hydrogen peroxide. Some cell experiments also used Euk-134, a combined SOD and catalase mimetic.
• Main results:
  • Crocidolite-induced lung fibrosis was significantly lower in MCAT mice than in wild-type mice, as measured by microscopy, lung fibrosis score and lung collagen.
  • MCAT mice were significantly protected against bleomycin-induced fibrosis compared with wild-type mice.
  • In wild-type mice, asbestos increased apoptotic (CC-3 positive) cells at bronchoalveolar duct junctions from 0.8% (titanium dioxide) to 10% per 220 junction cells (p less than 0.05).
  • In asbestos-exposed mice, apoptotic cells were 3% of 220 junction cells in MCAT mice versus 10% in wild-type mice (p less than 0.05).
  • Cells that were both apoptotic and alveolar type II cells (CC-3 and SFTPC co-positive) were 4% of 220 junction cells in asbestos-exposed wild-type mice versus 3% in MCAT mice. This difference was not statistically significant.
  • Mitochondrial DNA damage in type II cells from wild-type mice was 2.6 after asbestos versus 1 after titanium dioxide (ratio of lesions/10kB frequency per fragment, p less than 0.05).
  • In asbestos-exposed mice, mitochondrial DNA damage in type II cells was 1.1 in MCAT mice versus 2.6 in wild-type mice (p less than 0.05).
  • In dish experiments, mitochondrial DNA damage caused by amosite asbestos or hydrogen peroxide in wild-type type II cells was completely blocked in type II cells from MCAT mice.
  • Euk-134 blocked amosite asbestos- and hydrogen peroxide-induced mitochondrial DNA damage and apoptosis in MLE-12 cells.
  • In wild-type type II cells, amosite asbestos raised mitochondrial reactive oxygen production about 4-fold over control (10,000 versus 40,000 MitoSox fluorescence units, p less than 0.05).
  • After asbestos, mitochondrial reactive oxygen production was 20,000 fluorescence units in type II cells from MCAT mice versus 40,000 in cells from wild-type mice (p less than 0.05).
• The authors' conclusion: The authors concluded that the reduced fibrosis, mitochondrial DNA damage and apoptosis in MCAT mice after asbestos or bleomycin suggest a role for mitochondrial hydrogen peroxide-induced mitochondrial DNA damage in alveolar epithelial cells in promoting lung fibrosis. They reasoned that limiting this damage may be a novel therapeutic target for pulmonary fibrosis.
• Limitations the authors state:
  • The studies did not exclude a role for mitochondrial catalase in other cell types involved in lung fibrosis, especially lung macrophages and fibroblasts.
  • The findings do not exclude a role for other antioxidant lung defenses.
  • The Mito-Sox assay cannot precisely identify which specific free radicals are present.
  • No mouse model reproduces all the features of idiopathic pulmonary fibrosis, so the authors used two fibrosis models.

Topics

Medical terms in this study, explained in our Lexicon: Fibrosis, Reactive Oxygen Species (ROS), Apoptosis, Crocidolite, Collagen, Immunohistochemistry, Pulmonary Fibrosis.

Related studies

• Mitochondria-targeted Ogg1 and aconitase-2 prevent oxidant-induced mitochondrial DNA damage in alveolar epithelial cells — S.J. Kim, P. Cheresh, et al. (2014)
• SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis — R.P. Jablonski, S.J. Kim, et al. (2017)
• The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis — S.J. Kim, P. Cheresh, et al. (2015)
• The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes — P. Cheresh, S.J. Kim, et al. (2020)
• Asbestos causes apoptosis in alveolar epithelial cells: role of iron-induced free radicals — A. Aljandali, H. Pollack, et al. (2001)
• SIRT3 Overexpression Ameliorates Asbestos-Induced Pulmonary Fibrosis, mt-DNA Damage, and Lung Fibrogenic Monocyte Recruitment — P. Cheresh, S.J. Kim, et al. (2021)

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